Project Abstract: Dendritic cell (DC) antigen uptake and presentation is a crucial initial step in establishing adaptive immune responses. This project will characterize a small sub-set of DCs, called hyper-responder DCs, which display enhanced antigen uptake, immune activation and potentially initiate immune responses using paracrine signaling. We will first identify these DCs by observing their uptake of fluorescently labeled, polystyrene coated microparticles, which display toll-like-receptor (TLR) agonist molecules on their surface, and the kinetics of hyper-responder immune activation. Next, we will establish a transcriptional profile for hyper-responders by performing both bulk and single cell mRNA sequencing. By inhibiting cytokine release using the inhibitor brefeldin A, we will also assess the contribution hyper-responders make to the overall immune response via paracrine signaling and any specific genes regulated in a paracrine fashion. We will also repeat this analysis using synergistic combination of TLR agonists in order to identify genes responsible for TLR synergy. Any genes of interest will be validated through T cell interaction studies, western blots, flow cytometery and/or immunofluorescent staining. By characterizing the hyper-responsive cells and their transcriptional responses to paracrine signaling and TLR synergy, this project will advance our understanding of antigen presentation and aid in the development of more efficacious vaccines.